Tissue inhibitors of metalloproteinases (TIMPs) inhibit metalloproteinases, a multigene family of endopeptide hydrolases. The metalloproteinases are secreted by connective tissue and hematopoietic cells, use Zn.sup.++ or Ca.sup.++ for catalysis, and may be inactivated by metal chelators as well as TIMP molecules.
The matrix metalloproteinases (MMPs) participate in a variety of biologically important processes including the degradation of many structural components of tissues, particularly the extracellular matrix (ECM). This ability is desirable in processes where destruction of existing tissues is necessary, eg, in embryo implantation (Reponen P et al. (1995) Dev Dyn 202: 388-96), embryogenesis, and tissue remodeling. Metalloproteinases have also been implicated in processes where their activity is more specifically directed, such as in the movement of cells through tissues. Some of these molecules and their known substrates as reviewed in Murphy G and A J P Docherty (1992; Am J Respir Cell Mol Biol 120-125) are summarized below:
______________________________________ MMP-1 Interstitial Fibrillar Type X Collagens; Gelatin; collagenase Proteoglycan MMP-2 Gelatinase A Collagens IV, V, VII, X, XI; Elastin MMP-3 Stromelysin-1 Proteoglycan; Collagens II, IV, IX, X, XI; Fibronectin; Procollagen; Laminin; Gelatin; Collagenase; Gelatinase B MMP-7 Matrilysin Proteoglycan; Collagens II, IV, IX, X, XI; Fibronectin; Procollagen; Laminin; Gelatin; Collagenase; Gelatinase B; Elastin MMP-8 Neutrophil Fibrillar Collagens; Gelatin, collagenase Proteoglycans MMP-9 Gelatinase B Collagens IV, V, VII, X, XI; Elastin MMP-10 Stromelysin-2 Proteoglycan; Collagens II, IV, IX, X, XI; Fibronectin; Procollagen; Laminin; Gelatin; Collagenase; Gelatinase B ______________________________________
Metalloproteinases also inactivate several members of a class of serine protease inhibitors known as serpins. The inactivation of the serpins such as alpha-1 protease inhibitor allows serine proteases to destroy a variety of biologically important, non-ECM, molecules such as alpha-1 antitrypsin (Sorsa T et al. (1993) Agents Actions Suppl 39: 225-9). Therefore, controlling the activity of the metalloproteinases can have indirect effects on the activity of a range of other potent proteases.
It is, however, the MMPs which play the most important roles in pathological processes. In rheumatoid and other forms of arthritis, proteolysis of extracellular matrix components by MMPs is a major cause of cartilage and synovial tissue destruction (Firestein G S (1992) Curr Opin Rheumatol 4: 348-54). Similarly, the activity of elastinolytic MMPs secreted by mononuclear phagocytes has been implicated in the destruction of alveolar structure in pulmonary emphysema (Shapiro S D (1994) Am J Respir Crit Care Med).
MMPs are also associated with tumor metastasis. In order to colonize secondary sites, the primary tumor cells must both enter and exit the vascular system. This movement through tissues and the endothelial basement membrane is aided by the activity of metalloproteinases secreted by the tumor cells. In addition, the degradation of normal extracellular matrix structure is critical for angiogenesis as well as tumor cell migration (Ray J M and Stetler-Stevenson W G (1994) Eur Respir J 7: 2062-72; Mignatti P and Rifkin DB (1993) Physiol Rev 73: 161-95).
MMPs have also been implicated in periodontal disease where inflammation of the periodontium leads to connective tissue degradation and eventually to tooth loss. Tissue degradation is largely mediated by neutrophils which are attracted to sites of inflammation and secrete the MMPs believed to play a major role in tissue destruction (Sodek J and Overall C M (1992) Matrix Suppl 1: 352-62). MMPs are also involved in corneal ulcer formation following alkali burns and bacterial inflammation (Wentworth J S et al. (1992) 33: 2174-9; Burns F R et al. (1992) Matrix Suppl 1: 317-318).
MMPs participate in both normal and abnormal bone resorption. Coliagenase produced by osteoblasts degrades collagen on the surface of the bone and affords osteoclasts access to underlying mineralized bone. The osteoclasts utilize MMPs and other proteases to resorb bone. When catabolism exceeds deposition, the resulting imbalance can lead to demineralization of the bone and osteoporosis or osteoarthritis (Hembry R M et al (1995) Ann Rheum Dis 54: 25-32; Everts V et al. (1992) J Cell Physiol 150: 221-231; Vaes G et al. (1992) Matrix Suppl 1: 383-388). Even though TIMPs are expressed in osteoarthritic joints, the amount of inhibitory activity does not compensate for the amounts of MMPs carrying out degradation.
The involvement of MMPs in a wide range of pathological conditions suggests that natural inhibitors of MMPs, such as TIMPs, would be therapeutically useful for treatment of pathological conditions associated with excessive expression of MMPs.
TIMP Molecules
The nucleotide and amino acid sequences of three human TIMPs have been previously characterized and named TIMP-1 (Docherty A J P et al. (1985) Nature 318: 66-69), TIMP-2 (Boone T C et al. (1990) Proc Natl Acad Sci 87: 2800-2804; Stetler-Stevenson W G et al. (1990) J Biol Chem 265: 13933-38), and TIMP-3 (Wilde C G et al. (1994) DNA Cell Biol 13: 711-18). These proteins are classified as TIMPs based on their ability to inhibit metalloproteinases, structural similarity to each other, the 12 cysteines which form disulfide bonds important in secondary structure, and the presence of the VIRAK motif which interacts with the metal ion of the metalloproteinases.
Although human TIMPs inhibit a variety of metalloproteinases, the expression and specific activity of individual TIMPs do differ. TIMP-1, a 30 kD protein, is the most commonly expressed molecule and contains two asparagine residues which act as carbohydrate binding sites, one in loop 1 and one in loop 2 (Murphy and Docherty, supra). In addition, a truncated form of TIMP-1 which contains only the first three loops of the molecule is able to inhibit MMPs. Although TIMP-1 is a better inhibitor of interstitial collagenase than TIMP-2 (Howard E W et al. (1991) J Biol Chem 266: 13070-75), the 23 kD TIMP-2 molecule is the most effective inhibitor of gelatinases A and B. TIMP-3 is a 21 kD protein which inhibits collagenase 1, stromelysin, and gelatinases A and B (Apte S S et al. (1995) J Biol Chem 270: 14313-18) and may be induced by mitogens (Wick et al. (1994) J Biol Chem 269: 18953-60).
There have been reports of other inhibitors of metalloproteinases (IMPs) with physical characteristics different from those of the known TIMPs. In some cases, these activities result from alternate forms of the known TIMPs. For example, one IMP present in the conditioned media of a human bladder carcinoma was identified as a partially glycosylated form of TIMP-1, and another, as a partially processed/degraded form of TIMP-2 (Miyazaki K et al. (1993) J Biol Chem 268: 14387-93). Additional reports have described sources and characteristics of IMP activity, but active molecules have not been identified (Apodaca G et al. (1990) Cancer Research 50: 2322-29).
TIMPs which have been cloned from other species include bovine TIMP-1 (Freudenstein R et al. (1990) Biochem Biophys Res Comm 171: 250-256) and TIMP-2 (Boone T C et al. (1990) Proc Natl Acad Sci 87: 2800-2804); murine TIMP-1 (Gewert D R et al. (1987) EMBO J 6: 651-657); rabbit TIMP-1 (Horowitz S et al. (1989) J Biol Chem 264: 7092-7095); and chicken TIMP-3 (Pavloff N et al. (1992) J Biol Chem 267: 17321-6).